Correlation between positron annihilation lifetime and photoluminescence measurements for calcined Hydroxyapatite

Hydroxyapatite (HAp) Ca10(PO4)6(OH)2 is a compound that has stable chemical properties, composition, and an affinity for human bone. As a result, it can be used in odontology, cancer treatment, and orthopedic grafts to repair damaged bone. To produce calcined HAp at 600 °C with different pH values, a wet chemical precipitation method was employed. All synthesized HAp samples were characterized by X-ray diffraction (XRD), Raman spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), photoluminescence (PL), Zeta potential, and positron annihilation lifetime spectroscopy (PALS). The XRD results revealed that all calcined HAp samples were formed in a hexagonal structure with a preferred (002) orientation at different pH values. The crystal size of the samples was determined using the Scherrer equation, which ranged from 16 to 25 nm. The SEM and TEM results showed that the morphology of the samples varied from nanorods to nanospheres and rice-like structures depending on the pH value of the sample. The PL measurements indicated that the blue and green emission peaks of HAp were due to defects (bulk, surface, and interface) in the samples, which created additional energy levels within the band gap. According to Zeta potential measurements, the charge carrier changed from a positive to negative value, ranging from 3.94 mV to − 2.95 mV. PALS was used to understand the relationship between the defects and the photoluminescence (PL) properties of HAp. Our results suggest that HAp nanoparticles have excellent potential for developing non-toxic biomedical and optical devices for phototherapy.


Materials and methodology
The wet chemical precipitation method was used to create HAp samples at room temperature with varying pH levels.As shown in Fig. 1, the starting precursors were calcium nitrate tetrahydrate [Ca(NO 3 ) 2 •4H 2 O] (99%, Sigma Aldrich) and di-ammonium hydrogen phosphate [(NH 4 )2HPO 4 ] (98%, Sigma Aldrich), with the pH of the solutions adjusted by adding ammonium hydroxide [NH 4 OH] (Junsei Chem.Ltd., Japan).Deionized water was used as a solvent.Two groups were synthesized through chemical precipitation-one in an acidic environment without the addition of NH 4 OH (final pH = 5), and the other in a basic environment with the addition of NH 4 OH (final pH = 7, 9 and 11).Additionally, to enhance their properties, the samples were annealed at 600 ºC.The chemical solution used in this experiment consisted of two substances: 0.1 M Ca(NO 3 ) 2 •4H 2 O and 0.06 M of [(NH 4 ) 2 HPO 4 ] 28,29 .The precursors were dissolved in separate beakers containing 100 ml of deionized water.The Calcium nitrate tetrahydrate [Ca(NO 3 ) 2 •4H 2 O] solution was added at a constant rate of 3ml/min to the phosphate solution and mixed completely at 30 ºC by stirring with a magnetic stirrer at 350 rpm.Ammonium hydroxide Figures 2a and b display the XRD pattern for the samples prepared by the wet precipitation method.These samples were synthesized with various pH values, and Fig. 2a shows the pattern before annealing while Fig. 2b shows the pattern after annealing.The synthesized samples at pH 5 exhibited brushite as the predominant phase, with card no.(JCPDS 01-072-0713).Additionally, some peaks of hydroxyapatite with card no.(JCPDS 09-0432) were also observed.The miller indices for these peaks were (12-1), ( 040), (14-1), ( 121), ( 150), (15-2), (170), and (062).It is worth noting that the brushite formation was kinetically favored in acidic pH levels 30 .The brushite phase exhibits narrow diffraction peaks at half maximum intensity, indicating strong crystallinity.In contrast, the hydroxyapatite phase displays broad peaks that appear upon increasing the pH value to 7, 9, and 11, respectively, in the same figure.The XRD pattern of annealed samples at 600ºC is shown in Fig. 2b.This pattern displayed high crystallinity, as evidenced by the narrow and sharp diffraction peaks.In comparison, Fig. 2a showed broader diffraction peaks.At the lowest pH level, heat treatment transformed the synthesized brushite to calcium pyrophosphate (JCPDS 09-0346).Recent investigations have suggested that calcium pyrophosphate and β-TCP could be a viable alternative to pure hydroxyapatite in clinical settings 31,32 .The main diffraction peaks of hydroxyapatite (JCPDS 09-0432) were observed in all samples at physiological and high pH levels with a preferred orientation (002), (211), (300), (202), (310), ( 222), (213), and (004) planes positioned at 25.59°, 28.355°, 31.75°,32.845°, 34.024°, 39.812°, 46.527°, 49.249°, and 52.944°, respectively, by raising the pH value to 7, 9, and 11.The XRD pattern revealed that the as-prepared HAp had a poor crystalline phase, which was improved by annealing, resulting in a single calcium phosphate phase at pH 11.The samples of our hydroxyapatite (HAp) displayed diffraction patterns that suggest either nanometric crystallite size or poor crystallinity, like in vivo bone tissue created in physiological and higher pH values 30 .The diffraction peaks could be accurately assigned to the pure hexagonal HAp phase (space group P6 3 /m), and the average lattice parameter of the prepared and calcined samples were calculated to be almost equal to the standard values, with a = b = 9.41 Å, and c = 6.91 Å, α = β = 90, and γ = 120.We used Scherrer's equation 34,35 to determine the average crystallite size.
( www.nature.com/scientificreports/ The average size of crystals in nanometers is represented by D, while K is the constant value of the shape factor, which is fixed at 0.9.λ denotes the wavelength of radiation, β represents the full width at half-maximum (FWHM), and θ is the diffraction angle.Table 1 displays the measured average crystal size for both the asprepared samples and after annealing.
According to measurements, the particle size decreased as the pH values increased 36 .However, the particle size increased after annealing due to agglomeration caused by the heat treatment 37 .Therefore, the synthesis conditions heavily influence the production of hydroxyapatite (HAp) as a single phase or in conjunction with other orthophosphate phases 38 .

Morphological features
Figure 3a-d display the FE-SEM images taken to examine the morphology of the prepared samples with various pH values that were annealed at 600 °C. Figure 3a depicts the FE-SEM micrograph of the sample that was prepared in an acidic environment without the addition of NH 4 OH (pH5).It appears to have a nanorod shape.Increasing the pH value led to a reduction in particle size and changes in shape, which is supported by the XRD results.The shapes of the crystals changed from nano rods to nano wires, and agglomeration of nano rice-like structures.It is known that the initial pH levels affect the ion balance in the solution, which in turn influences the concentration of OH − , Ca 2+ , PO 4 3− , and (HPO 4 ) 2− .In Fig. 3b, for higher pH values equal to 7, the presence of the hydroxyl group in the solution during the growth of the Hap crystal affects the form of the crystal's planes a, b, and its growth direction (C axis).In Fig. 3c, the sample with a pH of 9.00 showed slow OH-Ca 6 formation on the a and b axes, but Ca 2+ was sufficient on the c axis to generate Ca-P 6 O 24 , resulting in HAp nanowires 39 .In Fig. 3d, it can be observed that increasing the pH level to 11 inhibits the formation of HAp, causing it to precipitate or The average crystal size for HAp samples at various pH, before annealing and after annealing.www.nature.com/scientificreports/form solid phases.This reduces its availability for chemical bonding due to the changes in solution equilibrium.At pH 11, the form of HAp changes to agglomerated rice grain-like structures because of the high concentration of hydroxide ions, which prevent extension 28,40 .

Transmission electron microscope (TEM) results
Figure 4a-c show TEM images of hydroxyapatite sample annealed at 600ºC with a pH of 11.As seen in Fig. 4a and b the sample consists of agglomerations of spherical and rice-like nanostructures, caused by the high concentration of hydroxide ions (OH -) at high pH values.This led to a rapid growth of the a, b, and c axes at the same speed due to the rapid attraction between the negative charge of the HAp nucleus and the positive charge of Ca-OH 6 39,41 .As a result of increased solubility, inhibition occurred, causing changes in the chemical equilibrium.This was due to the abundance of hydroxide ions (OH -) which greatly influenced the growth of primary unit particles and led to saturation of the solution at high pH values [42][43][44] .In Fig. 4c, the particle size distribution is shown to range from 16 to 30 nm, with an average particle size of 22.8 nm, highlighting its fabrication in nano size.

Raman spectroscopy results
Figure 5a and b show the Raman spectra of prepared samples that were created with different pH levels before and after annealing.In Fig. 5a, the Raman spectroscopy of brushite crystals (CaHPO 4 .2H 2 O) synthesized using wet chemical precipitation at pH 5 exhibited a distinct peak at 885 cm -1 , which corresponds to the phosphate group (P-O(H)).This is different from the Raman spectra of other samples 45 .On the other hand, the Raman spectrum of HAp at pH 7, 9, and 11 reveals a characteristic tetragonal PO 4 3− (v 1 ) group peak at 987 cm −1 , the PO www.nature.com/scientificreports/463, 593, and 606 cm −1 , which correlates to the ν 2 and ν 4 O-P-O.The bands that appeared at 966, 1045, and 1096 cm −1 correspond to v 1 symmetric and v 3 asymmetric stretching modes of P-O 47,48 .It has been found that certain organic materials exhibit bands at 1555 and 1650 cm −1 , which can be eliminated through the process of annealing 49,50 .Through annealing, all HAp samples bands were detected, which became more pronounced and narrower with increasing temperature.This indicates that the presence of phosphate ions increases, and HAp crystallization occurs at higher temperatures.The hydroxyl group's symmetric, stretching mode can be observed at 3570 and 629 cm −1 .The peaks of the phosphate ions (PO 4 ) 3− accurately appear between 961 and 964 cm −1 , which is the most intense peak.The of bands around 1045-1049 cm −1 is attributed to the phosphate vibrations 50,51 .These vibrations involve the symmetric, stretching mode of the phosphate group, indicating the presence of the hydroxyapatite crystalline structure.The bands of carbonate ions at 1475 cm −1 could be due to atmospheric adsorption 52,53 .

FTIR results
Figure 6 depicts the FTIR spectra of the HAp sample synthesized with pH11 and annealed at 600ºC.The adsorption bands that appeared between 570-610, 962-1052, and 1092cm −1 signify the symmetric bending, symmetric stretching, and asymmetric stretching vibrations of the phosphate (PO 4 ) 3− groups, respectively.The peak at 877 cm −1 represents the P-O bending vibration for (PO 4 ) 3− group, as stated in 28 .Additionally, there are three more peaks at 1410, 1457 and 1649 cm −1 which represent the stretching vibrations of C-O, C-O and C=O of the carbonate ions respectively.The minor peaks centered at 2000 and 2900 cm −1 show the triple bond C≡C and the C-H stretching vibrations of impurities found in the sample 54 . .The peaks observed at 630, 3443, and 3572 cm -1 indicate the bending and stretching vibrations of the hydroxyl group (OH) −28 .These findings authenticate the existence of hydroxyapatite fingerprint patterns, which confirms the presence of the hydroxyapatite phase in the synthesized samples at pH11 after annealing.These results agree with the Raman results.

Photoluminescence spectroscopy
this study, the photoluminescence spectra of calcium hydroxyapatite samples were observed at different pH levels (5, 7, 9, and 11) before and after annealing at 600 °C as shown in Fig. 7a and b.The highest photoluminescence peak was observed at around 400 and 515 nm.The sample analysis in Fig. 7a revealed that the photoluminescence intensity decreased with an increase in pH value (7, 9, and 11) in.This decrease could be attributed to www.nature.com/scientificreports/ the lower concentration of impurity luminescence centers (CO 3 2− ) in the samples.This finding is supported by previous studies 26,55 .After annealing, the luminescence behavior of identical samples was reversed, as shown in Fig. 7b.This result is attributed to the effect of pH value, as the spherical morphology enhances luminescence and brightness due to its high backing density and reduced light scattering 56 .
Additionally, the presence of nearby OH − groups can increase the number of interstitial vacancies.The decomposition of (CO 3 ) 2− and its substitutes can also create new energy levels within the forbidden zone 2,26 .These levels, which may be either deep or shallow, are caused by surface and bulk defects at the interface 2,16 .This leads to broadening in the PL spectra during annealing.Therefore, heat treatment can alter the order and disorder of the structure, resulting in an abundance of vacancies 26 .

Positron annihilation spectroscopy results
The positron lifetime spectra of the annealed hydroxyapatite samples can be separated into two components: an intermediate-lived component (τ 1 ) and a long-lived component (τ 2 ) with intensities I 1 and I 2 , respectively 57,58 .The τ 1 component is attributed to the annihilation of free positrons and para-positronium (p-Ps), while τ 2 component is attributed to the annihilation of ortho-positronium (o-Ps) pick-off annihilation, which depends on the size of the free volume defect.Figure 8a and b illustrate how τ 1 and I 1 change with varying pH values.The range of τ1 varies from (0.4106 ± 0.00083) to (0.41808 ± 0.00057), while I1 ranges from (97.325 ± 0.031) to (96.303 ± 0.030).For pH values of 5 and 7, τ 1 remains almost constant.However, when the pH value is increased to 11, τ 1 increases to 0.418 ns.This increase is likely due to the higher concentration of hydroxyl groups, which increase hydrogen bonding and enhance crosslinking.As a result, the electron density decreases and τ 1 increases while I 1 decreases.The decrease in I 1 may be a result of defect agglomeration, which increases the size of defects and decreases their concentration [59][60][61][62] .In Fig. 8c and d, you can see how τ 2 and I 2 change with varying pH values.The results indicate that τ 2 ranges from 2.67 to 3.01 ns, while I 2 ranges from 2.7 to 3.9%.For samples with pH values of 5 and 7, τ 2 decreased from 2.66 to 2.45ns, which may be due to incomplete synthesis and impurities of HAp 39,55 .When the pH level was increased to 11, τ 2 (tau 2 ) increased from 2.49 to 3.01 ns.This may have been caused by the formation of larger free volumes resulting from the aggregation of smaller free volumes, which grew due to the increase in pH level 63 .It has been observed that the values of I 2 have increased for all the samples of different pH levels, as shown in Fig. 8d.This increase in I 2 can be attributed to several reasons, one of which could be the strong oxidation caused by the heat treatment 64,65 .It is suggested that O 2 -radicals must move within the apatitic structure, causing the vacancy site to migrate in the crystalline material.This behavior is similar to that of TiO 2 , as reported by Okamoto et al.It is assumed that changes in the apatitic structure due to the removal of structural water at 900-1150ºC are influenced by the movement of holes 66,67 .
It is possible for I 2 to increase due to heat treatment, which removes water and results in the formation of an oxo bond with a lower electron density than the hydrogen bond 68,69 .Conversely, annealing can lead to the creation of hydroxyl ion vacancies (V OH ) due to the large number of OH -groups, which causes the strength of the signal to increase as the concentration of V OH grows 70 .It is also possible that a small amount of hydroxyl group may still be present even after heat treatment.This can act as an effective cation scavenger, boosting the synthesis of positronium by scavenging holes when introduced 71 .

Zeta potential results
Figure 9a and b display the ZP data for samples prepared with different pH values before and after annealing at 600 °C.In Fig. 9a, before annealing, the surface charge of the pH5 sample was negative (− 5.37 mv).However, as the pH value was increased, the charge carrier reversed and became positive, indicating that the sample underwent a hydrolysis process.This is because phosphate ions undergo the highest hydrolysis at low pH values [72][73][74] .The conversion of (PO 4 ) 3-into HPO 4 2− and H 2 PO 4 − is the probable source of the negative charge.This is supported by both Fig. 9a and Table 2. Increasing the pH value beyond 5 results in a positive surface charge.This is due to the high concentration of calcium ions.After annealing, the surface charge values, In Fig. 9b were all found to be negative.This is believed to be due to the presence of OH -groups, which ionize to O 2 − and H +75 .Additionally, both PO 4 3− and OH − groups are oriented on the outer surface of the Hap unit cell 76 .It's possible that particle morphology changes 77 , the presence of carbonate groups 78 , oxygen vacancies, and reversed hydrolysis could cause dehydration of the sample, resulting in the negative surface charge.By increasing the pH value, the  www.nature.com/scientificreports/negative surface charge of the particle decreases 79 .This decrease is believed to be caused by the dissociation of a functional group or the differential adsorption of ions from the solution on the particle's surface 80 .The sample with pH11 had the lowest negative ZP value (− 2.95 mV).This corresponds to a decrease in surface charge due to a high density of defects, which are responsible for emitting light signals and broadening the PL spectrum.These findings were confirmed by PALS, PL, and ZP measurements.Negative ZPs are beneficial in biological applications as they aid in Ca 2+ ion adsorption, promoting extracellular matrix for cell adhesion.In contrast, nanoparticles with positive surface charges are considered more toxic 81 .

Conclusion
HAp nanoparticles were successfully produced through the wet chemical precipitation method by annealing at 600°C at various pH levels.The crystal structure of the HAp nanoparticles was hexagonal, with a preferred orientation of (002) and ( 211), and a crystal size ranging from 16.3 to 25.23 nm.The shape of the nanoparticles varied with different pH values, ranging from nanorods to nano rice and spherical structures.The SEM and TEM images confirmed the shape variation.The PL spectra of the samples produced showed broad band profiles, with the highest emissions occurring in the blue and green regions of the visible electromagnetic spectrum at approximately 515 nm.This behavior was caused by the presence of bulk, surface, and interface defects, which resulted in a large density of intermediate energy levels (both shallow and deep) in the forbidden band.The results of Zeta potential analysis indicated a change in charge carriers caused by variations in pH during annealing at 600°C.Additionally, the results of PALS demonstrated that the HAp sample with pH11 annealed at 600°C had the highest oxygen vacancy defects.These defects create charge carriers, trapping centers, and emit light signals, contributing to an increase in the photoluminescence effect.Our findings indicate that the hexagonal HAp fabricated with pH11 annealed at 600°C is the most defective structure, which enhances the photoluminescence effect.Therefore, it can be an excellent material for phototherapy to track tumor cells.

Figure 1 .
Figure 1.Schematic diagram shows the hydroxyapatite preparation by using chemical percepitation method.

Figure 4 .
Figure5a and bshow the Raman spectra of prepared samples that were created with different pH levels before and after annealing.In Fig.5a, the Raman spectroscopy of brushite crystals (CaHPO 4 .2H 2 O) synthesized using wet chemical precipitation at pH 5 exhibited a distinct peak at 885 cm -1 , which corresponds to the phosphate group (P-O(H)).This is different from the Raman spectra of other samples45 .On the other hand, the Raman spectrum of HAp at pH 7, 9, and 11 reveals a characteristic tetragonal PO 43− (v 1 ) group peak at 987 cm −1 , the PO 4 3− (ν 2 ) vibrational mod O−P−O bending modes at 430 cm −1 .The bands at 1065, 1085, and 1151 cm −1 are due to the asymmetric stretching of P−O(ν 3 ), which indicates the existence of a disordered phosphate lattice in apatite.The phases of Brushite and HAp have different Raman spectra, showing distinct shifts in PO 43− .These shifts appear at 413 and 587 cm -1 and represent the symmetric bending mode's ν 2 and ν 4 .Additionally, the hydrogen phosphate anions found in synthetic brushite have vibrational modes and bands at 526 and 542 cm −1 .The stretching mode of the water molecule is indicated by the bands at 3276 and 3471 cm −146 .The HPO42-molecule's antisymmetric stretching mode (ν 3 ) is represented by the bands at 1118 and 1137 cm -1 .In Fig.5b, the synthesis of pure Hap was completed at pHs 7, 9, and 11.The functional bands of (PO 4 ) 3-, hydroxyl (OH -) appeared at

Figure 5 .
Figure 5. Shows the Raman spectrum of HAp (a) before annealing and (b) after annealing.

Figure 8 .
Figure 8.The variation of PAL components as a function of pH value of the measured samples: (a) τ 1 ns; (b) I 1 %; (c) τ 2 ns and (d) I 2 %).

Figure 9 .
Figure 9. Zeta Potential Spectrum of HAp as a function of pH value (a) before annealing (b) after annealing.

Table 2 .
Zeta potential values for HAp samples with different pH values before and after calcination.